Transcript Bacterial Source Tracking (BST)

The New Scientific
Advancement
Bacterial Source Tracking
(BST)
Overview
• Section 1- The Current Status
• Section 2 – Scientific Advancement:
Bacterial Source Tracking
(BST)
• Conclusion
• Discussion
Section 1
• Faecal contamination of water is a problem throughout
the world due to human health and safety concerns
• Impacts caused by harvesting closures are one of the
main impediments to shellfish industry development
• For example: Table 1: Number of New Closures and
Re-Opened Closure Orders for BC…In 2000, approx
105,000 ha closed to harvesting
Year
No. of New
Closures
No. of Closures
Revoked
(Re-opened)
1991 1992 1993 1994 1995 1996 1997 1998 1999 Total
7
13
12
4
30
5
4
15
25
4
105
5
3
2
2
2
18
Source: EC, 2001.
The New Scientific
Advancement
Bacterial Source Tracking
(BST)
Overview
• Section 1- The Current Status
• Section 2 – Scientific Advancement:
Bacterial Source Tracking
(BST)
• Conclusion
• Discussion
Section 1
• Faecal contamination of water is a problem throughout the
world due to human health and safety concerns
• Impacts caused by harvesting closures are one of the main
impediments to shellfish industry development
• For example: Table 1: Number of New Closures and Re-Opened
Closure Orders for BC…In 2000, approx 105,000 ha closed to
harvesting
Year
No. of New
Closures
No. of Closures
Revoked
(Re-opened)
1991 1992 1993 1994 1995 1996 1997 1998 1999 Total
7
13
12
4
30
5
4
15
25
4
105
5
3
2
2
2
18
Source: EC, 2001.
Section 1 Continued
• In Canada/US, monitoring programs use “indicator”
species- so called because their presence indicates that
faecal contamination may have occurred
• The MTF five tube dilution test has been used for more
than 60 yrs as the indicator test…but can/will never
address one of the major questions
• What is the source of the contamination?
• New methods called BST are under development to address
this question.
– Knowing the source (CAUSE) rather than
monitoring the level of microbial pollution
(SYMPTOMS) was the driving force behind the
development of BST methodologies
Background: Faecal
Coliforms
“Indicator” Organisms (Coliforms)
• Difficulties in the detection and identification
of viruses and bacteria in the environment and
in food samples led to the use of indicator
organisms.
What is Faecal Contamination?
• Faecal coliforms: bacteria (such as E. coli ) that live in
the digestive tract of all warm-blooded animals and are
excreted in the faeces have been chosen as the indicator
of the presence of disease-causing (pathogenic) viruses
and bacteria.
• Faecal coliforms: (generally)do not pose a danger to
people or animals, but indicate the presence of other
disease-causing bacteria and viruses, such as those that
cause typhoid and Hep A
Facts: Faecal Coliforms
• While E. coli is known to be faecal in origin,
others within this group, for example: Klebsiella
are found in soils and vegetation
• The non-faecal biotypes are frequently associated
with runoff, have a tendency to multiply in
nutrient-rich waters and give false-positives when
testing for faecal contaminated waters.
• This is a key issue for shellfish farmers as it
results in more frequent closures…and are
unnecessary
How Do Faecal Coliforms Get
into the Water?
• The transport of faecal coliforms to a water body
occurs either directly (point source) or indirectly
(non-point source)
• Directly = Point Source: refers to a single identifiable
source like a pipe
• Indirectly = Non-point sources: typically wetweather–dominated and diffuse in nature, in that they
do not enter water bodies from any single point (e.g.
urban litter, contaminated refuse, domestic pet/
wildlife excrement and failing sewer lines).
Limitations of the MTF Method
• 1) The MTF technique is not
E. coli specific. E. coli was
recommended to be used as the indicator organisms
instead of faecal coliforms because it gave a more
accurate measurement. So far, this is not a CSSP
standard.
• 2)Faecal coliforms have been isolated in pristine areas
• 3) Bacteria other than those that originated in the colon
can yield a positive faecal coliform test, for example,
Klebsiella
• 4) MTF testing is a slow process. People may become
sick before test results are released
• 5) MTF does not give specific information for source
identification.
Summary of Section 1
• Goals
– 1) Human health and safety issues – this is
imperative
– 2) Maximize certainty for growers (e.g.
cashflow, markets)
• But…the current system is only achieving
the first goal….and how well?
• Question – are there new tools available?
Section 2:
The New Scientific
Advancement
BST
BST Methods: 4 Types
• Molecular Methods (MST)
– Fingerprinting/Ribotyping
– Pulse Field Gel Electrophoresis
(PFGE)
– Randomly Amplified Polymorphic
DNA (RAPD)
• Biochemical Methods
(Phenotype)
• Species-Specific
Indicators (BST
tracking) *Note*: Not all
are listed
– Streptococcus bovis
– Clostridium perfringens
– Bacteroides fragilis group
– Rhodococcus coprophilus
– ß-glactosidase test
• Colilert Test: A Result of the ß –• Chemical BST
glactosidase method
Methods
– Multiple Antibiotic Resistance (MAR)
– Detergents/Optical
– Sterols or Fatty Acid Analysis
Brighteners
– Coliphages
– Fluorescent Dye Tracing
– Bacteroides
– Caffeine
– Coprostanol
Background: Molecular Methods:
(Microbial Source Tracking or MST)
• A bacteria’s genetic structure are clonal. That is, all descendants
of a common ancestral cell are genetically related to each other.
• Over time members of a clone may accumulate genetic changes:
diverge from the main group to form one or several new clonal
groups.
– The E. coli strain for example, that inhabits the intestines of
one species (e.g. humans) is genetically different from the
strain that might inhabit another (e.g. cows, dogs, deer or bear).
• MST makes use of this, in order to classify organisms based on
their genetic fingerprints into groups of clonal descent.
• When bacteria with an identical genetic fingerprint are isolated
from both a polluted site and a suspected animal source, the
species can be proven as a contributor
Background: Biochemical Methods
• Most of biochemical methods offer certain
advantages over molecular methods:
• less training for lab personnel
• lower/isolate cost
• potential to perform the methods on hundreds of
isolates per week (versus a few dozen isolates per
week, which is typical for molecular methods).
• Biochemical methods: based on the fact that an
organism’s genes actively produce a biochemical
substance
• The biochemical substances produced is what is
being measured.
Background: Species-specific
Indicators
• The extent to which faecal coliforms
settle, grow, and are re-suspended after
they are released into receiving waters
remains controversial, leaving most
indicator testing/accuracy in question.
• Testing whether there are better suited
alternate indicators than the feacal
coliforms though...remains relevant
Background: Chemical BST
Methods
• Chemical BST methods do not detect faecal
bacteria, but chemical compounds that are
associated with humans.
• These chemicals are often found in wastewater
such as septic tank effluent…if found in a
water body, then it is likely from a human
source
$$$ Costs $$$
 All US Funds
 Ribotyping analysis 5-8 isolates: $320.00 / sample
 MAR analysis of 20 E. coli isolates
$200.00 / sample
 Pulse field gel electrophoresis:
Genetic fingerprinting analysis of bacterial species.
2 restriction enzymes $180.00 /bacterial isolate
Conclusion
• There is no evidence that any one BST method will
emerge as the single best method for all situations
• Comparative test b/t the BST methods with the same
collection of isolates has yet to be done on a sufficiently
large scale.
• Obtaining similar results with different BST methods
may also improve the chances that the source
identifications are correct.
• A “toolbox” approach may seem warranted. 1st.. one
could save money using Biochemical or Chemical
methods. Then verify with MST afterward
• Therefore...a corroboration of results using different
methods
Conclusion
• Regulations are currently aimed at preventing faecal
contamination from any mammalian source
• BUT...Despite efforts to minimize faecal input into
coastal waterways a problem remains:
– The inability to identify the source
• It is also known that:
– the MTF technique will never address one of
the major questions that have perplexed water
quality managers for years…what is the source
of the contamination?
• Better-suited testing alternatives may be used to refine
closures while still protecting consumers
Conclusion
• BST Could be used as a tool for the ID and
remediation of upland sources of contamination
in sanitary surveys…. in advance of the validation
of techniques
• BST could be used as a preventative measure
instead of a mechanism to minimize or reduce
water contamination.
• BST = False positive prevention…e.g. Klebsiella
• In the meantime, as the costs of BST declines,
their use as a tool for prevention of contamination
may prove the best application
Conclusion
• The advantages provided by BST will
only be captured if there is a willingness
to adopt these new techniques into
regulation as they are validated
The End
Section 1 Continued
• In Canada/US, monitoring programs use “indicator”
species- so called because their presence indicates that
faecal contamination may have occurred
• The MTF five tube dilution test has been used for more
than 60 yrs as the indicator test…but can/will never
address one of the major questions
• What is the source of the contamination?
• New methods called BST are under development to address
this question.
– Knowing the source (CAUSE) rather than
monitoring the level of microbial pollution
(SYMPTOMS) was the driving force behind the
development of BST methodologies
Background: Faecal
Coliforms
“Indicator” Organisms (Coliforms)
• Difficulties in the detection and identification
of viruses and bacteria in the environment and
in food samples led to the use of indicator
organisms.
What is Faecal Contamination?
• Faecal coliforms: bacteria (such as E. coli ) that live in
the digestive tract of all warm-blooded animals and are
excreted in the faeces have been chosen as the indicator
of the presence of disease-causing (pathogenic) viruses
and bacteria.
• Faecal coliforms: (generally)do not pose a danger to
people or animals, but indicate the presence of other
disease-causing bacteria and viruses, such as those that
cause typhoid and Hep A
Facts: Faecal Coliforms
• While E. coli is known to be faecal in origin,
others within this group, for example: Klebsiella
are found in soils and vegetation
• The non-faecal biotypes are frequently associated
with runoff, have a tendency to multiply in
nutrient-rich waters and give false-positives when
testing for faecal contaminated waters.
• This is a key issue for shellfish farmers as it
results in more frequent closures…and are
unnecessary
How Do Faecal Coliforms Get
into the Water?
• The transport of faecal coliforms to a water
body occurs either directly (point source) or
indirectly (non-point source)
• Directly = Point Source: refers to a single
identifiable source like a pipe
• Indirectly = Non-point sources: typically wetweather–dominated and diffuse in nature, in
that they do not enter water bodies from any
single point (e.g. urban litter, contaminated
refuse, domestic pet/ wildlife excrement and
failing sewer lines).
Limitations of the MTF Method
• 1) The MTF technique is not
E. coli specific. E. coli was
recommended to be used as the indicator organisms
instead of faecal coliforms because it gave a more
accurate measurement. So far, this is not a CSSP
standard.
• 2)Faecal coliforms have been isolated in pristine areas
• 3) Bacteria other than those that originated in the colon
can yield a positive faecal coliform test, for example,
Klebsiella
• 4) MTF testing is a slow process. People may become
sick before test results are released
• 5) MTF does not give specific information for source
identification.
Summary of Section 1
• Goals
– 1) Human health and safety issues – this is
imperative
– 2) Maximize certainty for growers (e.g.
cashflow, markets)
• But…the current system is only achieving
the first goal….and how well?
• Question – are there new tools available?
Section 2:
The New Scientific
Advancement
BST
BST Methods: 4 Types
• Molecular Methods (MST)
– Fingerprinting/Ribotyping
– Pulse Field Gel Electrophoresis
(PFGE)
– Randomly Amplified Polymorphic
DNA (RAPD)
• Biochemical Methods
(Phenotype)
• Species-Specific
Indicators (BST
tracking) *Note*: Not all
are listed
– Streptococcus bovis
– Clostridium perfringens
– Bacteroides fragilis group
– Rhodococcus coprophilus
– ß-glactosidase test
• Chemical BST
• Colilert Test: A Result of the ß – Methods
glactosidase method
– Detergents/Optical
– Multiple Antibiotic Resistance (MAR)
Brighteners
– Sterols or Fatty Acid Analysis
– Fluorescent Dye Tracing
– Coliphages
– Caffeine
– Bacteroides
– Coprostanol
Background: Molecular Methods:
(Microbial Source Tracking or MST)
• A bacteria’s genetic structure are clonal. That is, all descendants
of a common ancestral cell are genetically related to each other.
• Over time members of a clone may accumulate genetic changes:
diverge from the main group to form one or several new clonal
groups.
– The E. coli strain for example, that inhabits the intestines of
one species (e.g. humans) is genetically different from the
strain that might inhabit another (e.g. cows, dogs, deer or bear).
• MST makes use of this, in order to classify organisms based on
their genetic fingerprints into groups of clonal descent.
• When bacteria with an identical genetic fingerprint are isolated
from both a polluted site and a suspected animal source, the
species can be proven as a contributor
Background: Biochemical Methods
• Most of biochemical methods offer certain
advantages over molecular methods:
• less training for lab personnel
• lower/isolate cost
• potential to perform the methods on hundreds of
isolates per week (versus a few dozen isolates per
week, which is typical for molecular methods).
• Biochemical methods: based on the fact that an
organism’s genes actively produce a biochemical
substance
• The biochemical substances produced is what is
being measured.
Background: Species-specific
Indicators
• The extent to which faecal coliforms
settle, grow, and are re-suspended after
they are released into receiving waters
remains controversial, leaving most
indicator testing/accuracy in question.
• Testing whether there are better suited
alternate indicators than the feacal
coliforms though...remains relevant
Background: Chemical BST
Methods
• Chemical BST methods do not detect faecal
bacteria, but chemical compounds that are
associated with humans.
• These chemicals are often found in wastewater
such as septic tank effluent…if found in a
water body, then it is likely from a human
source
$$$ Costs $$$
 All US Funds
 Ribotyping analysis 5-8 isolates: $320.00 / sample
 MAR analysis of 20 E. coli isolates
$200.00 / sample
 Pulse field gel electrophoresis:
Genetic fingerprinting analysis of bacterial species.
2 restriction enzymes $180.00 /bacterial isolate
Conclusion
• There is no evidence that any one BST method will
emerge as the single best method for all situations
• Comparative test b/t the BST methods with the same
collection of isolates has yet to be done on a sufficiently
large scale.
• Obtaining similar results with different BST methods
may also improve the chances that the source
identifications are correct.
• A “toolbox” approach may seem warranted. 1st.. one
could save money using Biochemical or Chemical
methods. Then verify with MST afterward
• Therefore...a corroboration of results using different
methods
Conclusion
• Regulations are currently aimed at preventing faecal
contamination from any mammalian source
• BUT...Despite efforts to minimize faecal input into
coastal waterways a problem remains:
– The inability to identify the source
• It is also known that:
– the MTF technique will never address one of
the major questions that have perplexed water
quality managers for years…what is the source
of the contamination?
• Better-suited testing alternatives may be used to refine
closures while still protecting consumers
Conclusion
• BST Could be used as a tool for the ID and
remediation of upland sources of contamination
in sanitary surveys…. in advance of the validation
of techniques
• BST could be used as a preventative measure
instead of a mechanism to minimize or reduce
water contamination.
• BST = False positive prevention…e.g. Klebsiella
• In the meantime, as the costs of BST declines,
their use as a tool for prevention of contamination
may prove the best application
Conclusion
• The advantages provided by BST will
only be captured if there is a willingness
to adopt these new techniques into
regulation as they are validated
The End